Discussion
Previous research (12) demonstrated that CRS could be subdivided into endotypes based on inflammatory markers. The division of CRS into endotypes is useful to understand the natural course of the disease and to make a treatment choice. Here, we examined the chemokine expression in the CRS tissues to cluster and match with the CRS phenotypes CRSwNP/ CRSsNP and the comorbidities asthma and N-ERD. To the best of our knowledge, this is the first study analyzing this panel of chemokines in the context of CRS. The results of the current study allow associating chemokines signatures to the endotypes mentioned above, with type 1-related biomarkers such as IP-10/ CXCL10 and TARC/ CCL17 as well as eotaxin/ CCL11 as early-type 2-related biomarkers. Additional neutrophilic and type 1-related inflammatory parameters were detected in IL-5 negative/ low and also in IL-5 high cluster groups, respectively.
Based on the chemokine expression pattern, we have identified seven CRS clusters in the dataset of the multicenter cooperation GA2LEN ”Chronic rhinosinusitis and nasal polyposis cohort study“(12). As analytical targets, we selected type-2-associated CC-chemokines and a neutrophil CXC-chemokine (Table 1 ). Previous studies revealed that the concentration of MIP-1α/ CCL3 (17), eotaxin/ CCL11 (18), TARC/CCL12 (19), and PARC/CCL18 (20) were higher in tissue samples obtained from CRSwNP patients, as opposed to the patients with CRSsNP. In addition, MCP-3/CCL7 was found to be increased in CRS patients but not providing distinction into CRSwNP and CRSsNP (21). Similarly, the concentration of IP-10/CXCL10 was reported to increase in response to viral infection in CRSwNP tissue (22). There was no difference reported in the levels of ENA-78/CCL5 between CRS patients and healthy subjects (23). However, none of these studies provided the classification of CRS endotypes, which we performed here using the expression of the type 2-cytokine IL-5 as basis for differentiation.
In the clusters 1-4, IL-5 levels were negative or low in the lowinflammatory or low mixed Th1/ Th2 inflammatory endotype groups. Corroborating the results of Tomassen et al. (12), we detected a low inflammatory pattern in cluster 1 with negative IL-5 levels. In cluster 2 with low IL-5 levels, we found elevated concentrations of a single chemokine (MCP-3/CCL7) in all patients who suffered from CRSsNP. The MCP-3/CCL7 receptor CCR1 is known to be expressed during Th1- and Th2 inflammatory responses, pointing to Th1 associated effects of MCP-3/CCL7 in cluster 2. In cluster 3 with mixed Th1/Th2 inflammatory patterns, eotaxin/CCL11 - a typical Th2-chemokine - and IP-10/ CXCL10 - a typical Th1- - were significantly elevated. Interestingly, in cluster-3, the levels of eotaxin/ CCL11 and TARC/CCL17 were significantly higher than in other clusters. At the same time, IL-5 and IgE concentrations showed no significant difference, suggesting a possibility for using eotaxin/ CCL11 and TARC/ CCL17 as early biomarkers of type-2 related inflammation. Eotaxin/ CCL11 serves as eosinophilic chemokine (24) for CRSwNP patients compared to controls (18, 25). TARC/ CCL17 is associated with a type-2-profile (19) and was found to be elevated in fibroblasts isolated from CRSwNP patients, following stimulation with IL-4 (26). In addition, its concentration was higher in nasal secretions from the CRSwNP compared to CRSsNP patients (27).
In clusters 5-7, we observed elevated concentrations of IL-5 in association with the type-2 inflammatory profile. In addition to TARC/ CCL17 and eotaxin/ CCL11, also the concentration of PARC/ CCL18 was significantly higher in these clusters. Type-2 cytokines upregulate PARC/ CCL18 in the sputum of asthma patients, and PARC/ CCL18 markedly correlates with the number of eosinophils in sputum (28). Elevated PARC levels were found in the CRSwNP tissues (20). PARC/ CCL18 and TARC/ CCL17 are considered to be typical chemokines produced by dendritic cells and are essential for the regulation of type-2 immune response as well as for trafficking of memory T cells, as described in atopic dermatitis (29). An increased number of dendritic cells were described in nasal polyps (30). In a study of Jonstam et al. the levels of PARC/ CCL18 and the eotaxin-group eotaxin-2 and -3 (CCL24/ CCL26) decreased significantly in nasal secretions following 16 weeks of therapy with dupilumab (31) and also reduced in serum in recent phase 3 studies in dupilumab versus placebo-treated CRSwNP patients (8).
Further, the concentration of MCP-3/ CCL7 and MIP-1α/ CCL3 (32) was significantly greater in the “high IL-5 group”, pointing at additional type-2 inflammatory effects of these chemokines. Both CC chemokines are known to chemoattract eosinophils in the type-2 inflammatory environment (33, 34). MCP-3/ CCL7 and MIP-1α/ CCL3 predominantly attract type-1/type-2 cells with MCP-3/ CCL7 attracting monocytes, dendritic cells, lymphocytes, NK cells, basophils, neutrophils, and eosinophils via CCR1 and CCR3 (35). In contrast, MIP-1α/ CCL3 attracts IFN−γ−activated neutrophils as well as a small subpopulation of CCR1-expressing eosinophils (33, 34).
Of interest, a typical marker of neutrophilic inflammation – MPO - was significantly elevated in all clusters, stressing the impact of neutrophiles in the not-Th2 CRSwNP with comorbid neutrophilic asthma, and also in the Th2 CRSwNP with eosinophilic asthma (35, 36). Similarly, the type-1 related cytokines IL-22and IFN−γ were overexpressed in low- and high-IL-5 clusters (clusters 1, 4, and 5; IFN−γ also in cluster 6). These results are comparable to the results of Tomassen et al. (12); however, in contrast to Tomassen et al., IL-17 and TNFα were only present in the low IL-5 cluster group. Yet, the relatively small number of patients per identified cluster in our study has to be considered a shortcoming. More studies should follow to confirm our results and add statistical weight to the findings.
Corroborating previous results (12), also in our study, the phenotype of CRSwNP correlated positively with the IL-5 concentrations, increasing from 11% in cluster-1 to 100% in cluster-7. This observation confirms recent data about CRSwNP subjects with a negative IL-5 profile, which is of importance to consider before making the therapeutic decision regarding use of type 2-related biologicals. Additionally, also non-type-2 asthma exists in CRSwNP subjects (9% in cluster-2), in contrast to the type-2-biased inflammatory patterns with an asthma prevalence of 50% in cluster-7. We diagnosed N-ERD only in type-2 endotypes, consistent with the results of Tomassen et al. The relevance of IFN-γ being present in N-ERD-patients was described before (37).
In conclusion, the endotype clustering of chronic rhinosinusitis based on chemokine expression pattern has confirmed and extended previous findings of CRS being a heterogeneous inflammatory disease (12). We identified detailed CRS endotypes characterized by a wide diversity of inflammatory markers. Increased levels of TARC/ CCL17, eotaxin/ CCL11, and PARC/ CCL18 associated with type-2 biomarkers in CRSwNP, and TARC and eotaxin may serve as early markers in patients without upregulation of IL-5 and IgE. Additional neutrophilic biomarkers contribute to endotype characterization in CRSsNP and CRSwNP. Our observations should be further expanded to increase the general knowledge about CRS, to augment the diagnostic and monitoring possibilities, and tune-up the therapeutic approach. The recent expansion of biologics drugs used to treat upper respiratory tract conditions fully justifies the individual, patient-directed diagnostic approach to warrant a successful treatment and monitoring of CRS.